Stereocomplexed microparticles loaded with Salvia cadmica Boiss. extracts for enhancement of immune response towards Helicobacter pylori

Controlled delivery of therapeutic substance gives numerous advantages (prevents degradation, improves uptake, sustains concentration, lowers side effects). To encapsulate Salvia cadmica extracts (root or aerial part), enriched with polyphenols with immunomodulatory activity, in stereocomplexed microparticles (sc-PLA), for using them to enhance the immune response towards gastric pathogen Helicobacter pylori. Microparticles were made of biodegradable poly(lactic acid) (PLA) and poly(d-lactic acid) (PDLA). Their stereocomplexation was used to form microspheres and enhance the stability of the obtained particles in acidic/basic pH. The release of Salvia cadmica extracts was done in different pH (5.5, 7.4 and 8.0). The obtained polymers are safe in vitro and in vivo (guinea pig model). The sc-PLA microparticles release of S. cadmica extracts in pH 5.5, 7.4, and 8.0. S. cadmica extracts enhanced the phagocytic activity of guinea pig bone marrow-derived macrophages, which was diminished by H. pylori, and neutralized H. pylori driven enhanced production of tumor necrosis factor (TNF)-α and interleukin (IL)-10. The sc-PLA encapsulated S. cadmica extracts can be recommended for further in vivo study in guinea pigs infected with H. pylori to confirm their ability to improve an immune response towards this pathogen.


Results and discussion
This work aimed to prepare biocompatible polymeric microparticles able to withstand the conditions in the colon. In this regard, stereocomplexed PLA microparticles are great candidates due to better hydrolytic stability than enantiomeric ones when the macromolecules are not functionalized with pH-responsive end groups. The first step was the synthesis of the desired PLLA and PDLA by ROP of lactide with the catalytic/initiating system composed of triflic acid and hexane-1,6-diol. Two sets of macromolecules were obtained: low molecular mass (lmm) with M n of 3000 g/mol and medium molecular mass (mmm) with M n of 7500 g/mol, as shown in Figs. S1-S3. These PLAs were subsequently used for the preparation of microparticles. The mixing of an equimolar amount of PLLA and PDLA induces stereocomplexation between polymeric chains, and as a result network composed of hydrogen bonds can be formed 7 . To achieve this aim, PLLA and PDLA were dissolved in THF (with or without studied S. cadmica extracts) to prepare stereocomplexed microparticles. Subsequently, Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) analysis was employed to prove the formation of stereocomplex assemblies Fig. S4. The band at 909 cm −1 appeared after mixing PLLA and PDLA, which is responsible for the helical conformation of the stereocomplex 37 . Subsequently, DSC can be used to assess the thermal properties of the obtained materials Figs. S5, and S6. This technique can provide information about the purity of the obtained stereocomplexed microparticles since it shows the melting of even a small amount of homocrystallites. Among all tested samples only for sc-PLA-SCAPE (lmm), the presence of a small amount of homocrystallites was observed in the first heating run of the differential scanning calorimetry (DSC). In the second heating run, the stereocomplex crystallites were observed exclusively. Moreover, the effect of molar mass on the stereocomplexed microparticles melting temperature (T m ) was observed, and those composed of medium molar mass exhibit T m approximately 20 °C higher than stereocomplexes obtained by mixing of low molecular mass PLLA and PDLA. In addition, the decomposition temperature (T d ) is also higher for stereocomplexed microparticles made of medium molar mass enantiomers. Interestingly, the T d of obtained microparticles increased after the addition of S. cadmica extracts (Tables S1, S2). This result confirmed that the presence of extracts improved a heat resistance of the resulting microparticles.
Scanning electron microscopy (SEM) is an excellent tool for acquiring information about the morphology of the obtained microparticles. The spherical morphology is typical for stereocomplexed microparticles, as shown in Fig. 1. All microparticles were porous with a visible amount of cracks. Interestingly, the diameter of the blank microparticles was close to 5 µm, whereas there was a high decrease in their size to 1-2 µm after the addition of S. cadmica extracts. This observed decrease in size can be related to the interactions of PLLA and PDLA macromolecules with plant extracts or the presence of dimethyl sulfoxide (DMSO) during their self-assembly. For instance, the encapsulation of doxorubicin (DOX) in stereocomplexed microparticles causes a decrease in www.nature.com/scientificreports/ particle size from µm to nm, as shown in our previous work 9 . However, the additional factor that may influence the self-assembly process is the addition of DMSO, which enhances the solubility of the extracts. Even a small amount of solvent such as DMSO may change the dynamics of H-bond formation 39 and the size of the resulting microparticles 40 . Therefore, these two substances are responsible for variation in the process of PLA macromolecule self-assembly since both can disrupt the formation of H-bonds.
In vitro validation of cellular effects of stereocomplexed microparticles unloaded or loaded with S. cadmica extracts. Bio-polymers for medical applications have had to meet cytocompatibility and genotoxicity criteria in vitro, even in the early stages of composite optimization (ISO 10993-5:2009). Our microparticles are made of enantiomeric PLA that can form stereocomplexed microparticles. PLA is a biocompatible and biodegradable polymer approved by Food and Drug Administration 41,42 . Thus, PLA has been explored regarding many therapeutic applications, including antigen and drug delivery vehicles [41][42][43] .
In this study we assessed the biocompatibility of sterecomplexed microparticles empty or loaded with S. cadmica extracts. For in vitro assessment of the biocompatibility of tested microparticles we used the reference L929 mouse fibroblasts, which are recommended by ISO standards; furthermore, Cavia porcellus-guinea pig fibroblasts, guinea pig primary gastric epithelial cells and human THP-1 monocytes. The biocompatibility of tested formulations was evaluated on the basis of 3-(4,5-dimethylthiazol-2-yl)-2,5-,diphenyltetrazolium bromide (MTT) reduction assay, signs of DNA damage and apoptosis. The results on the safety of the biomaterials are presented in Fig. 2.
The S. cadmica extracts (SCAPE and SCARE) alone do not affect the cell viability ( Fig. 2A). Similarly, stereocomplexed microparticles (sc-PLA), empty or loaded with S. cadmica extracts, were safe to L929 cells as well as guinea pig fibroblasts, guinea pig primary gastric epithelial cells and human THP-1 monocytes because the cell viability in MTT reduction assay was higher than 70% ( Fig. 2A).
To exclusion the potential risk of genotoxicity of the studied formulations we used guinea pig primary gastric epithelial cells and fibroblasts treated with S. cadmica extracts alone, empty microparticles alone or microparticles loaded with S. cadmica extracts: sc-PLA (lmm), sc-PLA (mmm), sc-PLA-SCAPE (lmm); sc-PLA-SCAPE (mmm); sc-PLA-SCRE (lmm); sc-PLA-SCRE (mmm). We assessed the presence of phosphorylated molecule gamma H2A.X (phospho-Ser139), which is induced in response to DNA double strand breaks and supported this procedure by staining cell nuclei with 4ʹ,6-diamidyno-2-fenyloindol (DAPI). We showed no increase in DNA damage in cells treated with S. cadmica extracts alone, microparticles alone or microparticles loaded with S. cadmica extracts as compared to control cells (Fig. 2B).
Our results are in line with the study developed by Uzun et al., excluding PLA genotoxicity using Chinese Hamster Ovary (CHO-K1) cell line by comet assay and cytokinesis-blocked micronucleus (CBMN) assay 44 .
Programmed cell death-apoptosis occurs during the development and ageing of cells, and provide maintaining global homeostasis in the host organism [44][45][46] . Upregulation of apoptosis can affect the equilibrium between cell growth and cell death, resulting in organ dysfunction 47 . We checked whether plant extracts and sc-PLA (lmm); sc-PLA (mmm); sc-PLA-SCAPE (lmm); sc-PLA-SCAPE (mmm); sc-PLA-SCRE (lmm) or sc-PLA-SCRE  www.nature.com/scientificreports/ (mmm) do not increase cell apoptosis. In four different assays, including: terminal deoxynucleotidyl transferase dUTP nick end labeling-TUNEL assay, estimation of caspase 3-CC3, caspase 9-CC9 and carbamoyl-phosphate synthetase 2 asparate transcarbamylase, and dihydroorotase-CAD none of tested S. cadmica extracts and sterecomplexed microparticles, unloaded or loaded with plant extracts showed pro-apoptotic activity (Fig. 2C). According to literature, PLA may induce inflammatory responses, due to its hydrophobicity 48 , and release of acidic degradation by-products 49,50 . In this study we evaluated the ability of S. cadmica extracts and stereocomplexed microparticles unloaded: sc-PLA (lmm), sc-PLA (mmm), sc-PLA-SCAPE (lmm) or loaded with S. cadmica extracts: sc-PLA-SCAPE (mmm); sc-PLA-SCRE (lmm); sc-PLA-SCRE (mmm) to activate in THP1-XBlue monocytes of nuclear factor kappa-B (NF-kappa B) signaling pathway, on the basis of the release of secretory embryonic alkaline phosphatase-SEAP (Fig. 3) www.nature.com/scientificreports/ tested samples vs. control cells sub-cultured in culture medium alone (negative control), while cells treated with lipopolysaccharide (LPS) Escherichia coli (positive control), which is recognized by toll-like receptor (TLR)-4 on NF-kappa B pathway, responded by the significant SEAP production. Strong activation of monocytes by the components of different biomaterials alone, biologically active substances used for encapsulation or contaminants such as endotoxin may induce acute inflammation, potentially resulting in pus formation, tissue degradation, and disintegration of cell barrier 51 . According to the Food and Drug Agency Guidance as well as the European Medicines Agency, an endotoxin content cannot be higher than 0.25 EU in biomaterials being in contact with human blood/tissue. Only controlled inflammation leads to revascularization and regeneration of injured tissue. S. cadmica extracts or microparticles used in this study did not show pro-inflammatory activity. This observed behavior could be attributed to the enhanced stability of stereocomplexed microparticles in vivo in comparison to enantiomeric components. It was shown 52 that degree of inflammatory reaction is correlated with the bulk size of sc-PLA nanofibers mats after implantation. The stereocomplexed nanofibers' morphology, crystallinity, and molar mass were unchanged after 12 weeks, whereas significant breakdown was observed for enantiomeric PLAs. Therefore, it can be anticipated that cross-linking of PLLA and PDLA chains suppresses the hydrolysis of molecular chains in vivo, and the inflammatory reaction is limited 52 . Activation of NF-kappa B was evaluated in cells incubated for 24 h with S. cadmica extract alone (root-SCRE or aerial part-SCAPE), stereocomplexed microparticles with low molecular mass-sc-PLA (lmm) or with medium molecular mass-sc-PLA (mmm), or such microparticles loaded with S. cadmica aerial part extractsc-PLA-SCAPE (lmm); sc-PLA-SCAPE (mmm) or root extract-sc-PLA-SCRE (lmm); sc-PLA-SCRE (mmm) on the basis of secreted embryonic alkaline phosphatase (SEAP). Cells in culture medium alone (Med) were used as a negative control, while monocytes stimulated with lipopolysaccharide (LPS) of E. coli (Ec) served as a positive control. Data are presented as median values ± range of four separate experiments (four independent experiments in triplicate for each experimental variant). Statistical significance: *p < 0.05; *untreated cells and cells treated with tested polymers vs. cells treated with LPS Ec.

Kinetics of S. cadmica extracts release from microparticles in different pH in vitro.
Due to the fact that sc-PLA-SCAPE (mmm) or sc-PLA-SCRE (mmm) loaded with S. cadmica extracts (aerial part-SCAPE or root-SCRE) were investigated in vivo, the same set of microparticles was used to elucidate the extracts release rate in acidic (pH 5.5), neutral (pH 7.2), and basic (pH 8.7) conditions. The experiments were conducted for 7 days. It was observed that the release rate ranges from 37 to 70%, as shown in Fig. 4. In general, the slowest release is observed in acidic pH, whereas in neutral and basic pH, it depends on the type of encapsulated extract. The observed increase in release rate for sc-PLA-SCAPE (mmm) microparticles (Fig. 4A) than sc-PLA-SCRE (Fig. 4B) may be attributed to the localization of a high amount of extract on the surface of the particles. This is correlated with the higher initial burst release from these microparticles, which is in agreement with the literature examples 32,53 . Moreover, the porosity of the obtained microparticles may play a role in the difference in extracts' release 54,55 .
It is worth noting that the most important time for extract release from microspheres is for 24 h or 48 h of their presence in the gut or small intestine. It can be observed that microspheres release 12.6% (acidic pH), 12.9% (neutral pH), and 19.5% (basic pH) of SCAPE after 24 h, whereas the amount of released SCRE was 10.8% (acidic pH), 14% (neutral pH), and 8.3% (basic pH). After 48 h of extracts release, the 21.1% (acidic pH), 22.6% (neutral pH), 30% (basic pH) for SCAPE and 18.9% (acidic pH), 22.5% (neutral pH), 20.2% (basic pH) for SCRE. It can be seen that there is only a slightly faster release of SCAPE; however, the difference is not significant. It can be anticipated that obtained microspheres would provide a prolonged release of extracts in the stomach 56 , due to the observed extracts release in vitro (Fig. 4). In addition, the size of obtained microparticles predisposes their uptake by a small intestine as it was shown by Reineke et al. for polystyrene particles 57 therefore, those particles may reach the desired target.  www.nature.com/scientificreports/ It was particularly interesting to see if extracts would be released at a pH around 8, which is found in the intestine, where interaction of plant compounds with the mucosal lymphatic system can be expected. On the other hand, the release of extracts at acidic pH in the stomach may facilitate the direct action of the plant formulation on bacteria in this milieu 27 . Both effects can be useful in controlling H. pylori infection. To confirm the assumed effects of the studied extracts, further in vivo studies will be developed on the guinea pig model.
In vivo biosafety of stereocomplexed microparticles unloaded or loaded with S. cadmica extracts. Particles made of PLA for potential medical applications should be characterized towards their interactions with the human tissues 58,59 . In this study, initial biosafety tests were performed on guinea pigs using stereocomplexed microparticles alone: sc-PLA (mmm) or such particles loaded with S. cadmica extracts: sc-PLA-SCAPE (mmm); sc-PLA-SCRE(mmm). For in vivo biosafety study the sc-PLA (mmm) were selected since  www.nature.com/scientificreports/ the encapsulation of S. cadmica extracts using these microparticles was better than using sc-PLA (lmm) microparticles. Subcutaneous injection of S. cadmica extracts, sc-PLA alone or sc-PLA loaded with S. cadmica extracts did not induce signs of skin irritation and edema or erythema in the vicinity of the site of injection during the course of the experiment, up to 72 h after subcutaneous injection (Fig. 5A). After subcutaneous injection of studied formulations selected tissues and organs: spleen and liver did not show signs of inflammation and splenocytes did not show an increased proliferative activity (Fig. 5B). The levels of aminotransferase (AST) or alanine aminotransferase (ALT) in the liver homogenates (Fig. 5Ca,b) and in serum samples (Fig. 5Cc,d) were similar in control animals and those injected with empty sc-PLA microparticles or such microparticles loaded with S. cadmica extracts. Similarly, there was no difference in the serum concentration of TNF-α and IL-1 B in control and tested animals.
Furthermore, testing of proliferation of splenocytes, the levels of ALT and AST in the liver homogenates and serum samples as well as serum level of TNF-α/IL-1 B were performed after per os inoculation of guinea pigs with sc-PLA (mmm), empty or loaded with S. cadmica extracts (Fig. 6). There was no difference between studied biomarkers in control animals vs. animals inoculated with the microparticles.
Influence of S. cadmica extracts on activity of guinea pig bonne marrow derived macrophages. Considering per os application of stereocomplexed microparticles loaded with S. cadmica extracts for increasing an immune response towards gastric pathogen H. pylori in this study we asked whether these extracts are able to increase an antibacterial activity of guinea pig bone marrow derived macrophages (BMDM). Macrophages are essential players in the maintenance of intestinal homeostasis and in stimulation of immunity in the gut 60 . The major activities of macrophages include the ability to engulf and digest infectious agents, present their antigenic determinants to T lymphocytes and to deliver proinflammatory cytokines 61 . The bone marrow derived macrophages are a good experimental model since they can differentiate into mature macrophages in vitro 61 . It has been shown that polyphenols, biologically active components, increased the number of T helper 1 (Th1) lymphocytes, natural killer (NK) cells, macrophages and dendritic cells (DCs) in Peyer's patches and spleen in C3H/HeN mice, after oral administration of polyphenols extracted from fruits 28 . In humans, the number of regulatory T lymphocytes can be boosted by polyphenols 29,30 . During H. pylori infection an inflammatory response in the gastric mucosa is elevated and becomes chronic, and due to this the potential therapeutic compound should be selected taking into account its pro-or anti-inflammatory activity. In this study we used S. cadmica aerial part and root extracts enriched in polyphenols to pulse BMDM for 24 h (induction), to see whether priming of cells with these plant extracts will result in upregulation of phagocytic activity of macrophages in conjunction with an increased expression of CD11b integrin, involved in phagocytosis, as well as secretion of proinflammatory TNF-α or anti-inflammatory IL-10. We also checked whether restimulation of cells with the same extract for 5 days will result in stronger macrophage response. Furthermore, we wanted to know how cells primed with S. cadmica extracts will react to restimulation with H. pylori. Finally, whether additional restimulation of cells for 24 h with S. cadmica extract will potentially neutralize an inhibitory effect driven by H. pylori. Previously we showed that H. pylori using their surface haemagglutinins and LPS inhibit phagocytosis 19,21 . Also Allen et al., showed that these bacteria may survive intracellularly in megasomes 20 . In this study we assessed the ability of guinea pig BMDM to engulf fluorescently labeled E. coli to see whether the ingestion can be upregulated in response to S. cadmica extracts.
Phagocytic activity of BMDM is demonstrated in Fig. 7A. Macrophages primed with S. cadmica aerial part or root extract (24 h), and then restimulated with the homologous extract for 5 days, showed an increased phagocytic activity towards E. coli (Fig. 7Ai,ii). The phagocytic activity of macrophages primed with S. cadmica extracts followed by restimulation of cells for 5 days with H. pylori was diminished (Fig. 7Ai,ii,iii). However, an additional restimulation of macrophages with S. cadmica extract for 24 h neutralized the negative effect driven by H. pylori (Fig. 7Aiv). Also other researchers have shown that plant extracts can enhance the phagocytosis process, which is consistent with results obtained in this study [62][63][64] .
Beneficial effect of S. cadmica extracts in the course of phagocytosis might be related to enhanced expression of macrophage CD11b adhesin molecules (Fig. 7B), which promote the interaction of macrophages with endothelial cells and function as complement receptors increasing complement dependent engulfment of infectious agents [65][66][67] . In the current study an increased expression of CD11b on BMDM was demonstrated 6 days after stimulation of cells with S. cadmica extracts (Fig. 7Bi,ii), which means that potentially also another factors may drive the enhancement of macrophage phagocytic activity in response to S. cadmica extracts. It is interesting that restimulation with H. pylori of cells, which were primed with S. cadmica extracts, resulted in diminished deposition of CD11b, which was compatible with diminished phagocytic activity of macrophages (Fig. 7Biii). However, additional restimulation of macrophages with S. cadmica extracts upregulated the expression of CD11b, which was related to elevated macrophage phagocytic activity (Fig. 7Biv).
Infection with H. pylori is correlated with the chronic inflammatory response in the gastric tissue. Inflammation is under control of pro-inflammatory and anti-inflammatory cytokines delivered by gastric epithelial cells, endothelium and immunocompetent cells, including macrophages. In this study we assessed whether S. cadmica extracts are able to neutralize proinflammatory cytokine-TNF-α, induced by H. pylori. We showed that S. cadmica extracts alone did not increase the production of TNF-α after priming of cells or priming of them and then restimulation (Fig. 7Ci,ii). However, S. cadmica extracts effectively diminished the production of this pro-inflammatory cytokine, which was upregulated in macrophages restimulated with H. pylori (Fig. 7iii,iv).
S. cadmica extracts induced the production of IL-10 by macrophages, which were exposed to plant formulation for 24 h and restimulated for 5 days with the homologous extract (Fig. 7Di,ii) www.nature.com/scientificreports/ www.nature.com/scientificreports/ of IL-10 production (Fig. 7Diii). In the previous study we showed that LPS H. pylori due to induction of intense IL-10 production by immune cells diminished the expansion and cytotoxic activity of natural killer cells, which together with granulocytes and macrophages consist the first line of natural immune defense 22 . In the current study the enhanced production of IL-10 by macrophages in response to H. pylori was diminished after an additional restimulation of cells for 24 h with S. cadmica extracts (Fig. 7iv). The immunomodulatory properties of S. cadmica extracts resulting in an enhancement of phagocytic activity of macrophages and diminishing of H. pylori driven TNF-α or IL-10 production potentially may help to control H. pylori infection as well as related chronic inflammatory response. Flavonoids present in the studied S. cadmica extracts might be responsible for diminishing H. pylori driven production of investigated cytokines by www.nature.com/scientificreports/ macrophages and elevated phagocytic activity. Similar effects of flavonoids present in different plant extracts have been shown by other researchers on various cell culture models in vitro [68][69][70][71][72] . Thus stereocomplexed microparticles (sc-PLA) loaded with S. cadmica extracts potentially may deliver their immunomodulatory components to the gut, where they can increase phagocytic activity of macrophages in conjunction with enhancement of CD11b expression as well as regulation of TNF-α and IL-10 secretion. Further in vivo study is needed on the guinea pig model, which was well characterized by us in terms of an immune response during H. pylori infection 73 , to see whether S. cadmica extracts delivered by sc-PLA will help to eliminate or prevent ongoing experimental infection with these bacteria, and control an inflammatory response.

Conclusions
In this study we developed method of encapsulation of S. cadmica extracts, enriched with polyphenols with immunomodulatory activity, in stereocomplexed microparticles (sc-PLA). We showed that obtained polymers are safe in vitro and in vivo. S. cadmica extracts enhanced the phagocytic activity of guinea pig bone marrowderived macrophages, which was diminished in response to H. pylori, and neutralized H. pylori-driven enhanced production of TNF-α and IL-10. Therefore, S. cadmica extracts encapsulated in sc-PLA can be recommended for further in vivo study to confirm their role in supporting the immune response towards H. pylori in guinea pigs experimentally infected with these bacteria.

Materials and methods
Chemicals. l,l-lactide and d,d-lactide (99%, Purac, Gorkum, Netherlands) were consecutively crystallized from dry 2-propanol and purified just before use by sublimation in vacuo (10 −3 mbar, 85 °C). Triflic acid (trifluorometanosulfonic acid, 98% Merck, Darmstadt, Germany) was used without further purification. Methylene chloride was distilled over P 2  Hydromethanolic (80% v:v) Salvia cadmica Boiss. (extracts derived from lyophilized and powdered roots and aerial parts of 6-month-old plants) was preparation and prepared their chemical characteristic by using highperformance liquid chromatography (HPLC) as previously described 74 . Detailed method of extracts preparation and their chemical characteristic have been described previously 74 .
All experiments on plants (either cultivated or wild), including the collection of plant material, were performed in accordance with the institutional, national, and international guidelines and legislation.
Polylactide synthesis. The PLLA and PDLA enantiomeric polymers were synthesized by ring-opening polymerization (ROP) of appropriate lactide 75 . L,L-LA (4.6 g, 31.9 mmol) was placed in a Schlenk tube and degassed under reduced pressure. After filling the reactive vessel with argon, methylene chloride (15 mL) was added via a syringe. After the complete dissolution of the lactide, hexane-1,6-diol (1.59 or 3.18 mmol) and trifluoromethanesulfonic acid (0.43 mmol) were introduced through the rubber septum. Polymerization was conducted at RT for 24 h; then a sample was withdrawn for SEC analysis. The remaining polymerization mixture was diluted with methylene chloride and CaO was added to neutralize trifluoromethanesulfonic acid. CaO was filtered off, the solution was precipitated in cold methanol, and the obtained product was dried under vacuum. The purified homopolymer was characterized by 1 H NMR. 1

Synthesis and characterization of microparticles and encapsulation of extracts.
The spontaneous precipitation was used to prepare stereocomplexed microparticles (MPs) 10 . To achieve this aim, 100 mg of PLLA and 100 mg of PDLA were dissolved separately in 4 mL of tetrahydrofuran (THF) and mixed without stirring. The interactions between enantiomeric chains lead to the precipitation of polymers in the form of microparticles. Finally, the supernatant was removed, the microparticles were washed with THF and dried under a vacuum. The sterocomplexed microparticles were loaded with the SCAPE or SCRE extract as follows: 100 mg of PLLA and 100 mg of PDLA were separately dissolved in 4 mL of THF. To dissolve 10 mg extracts the 10 mL water/ethanol (7:3) was required and 400 µL of the extract solution was added to the solution of one www.nature.com/scientificreports/ enantiomer along with 0.5 mL of dimethyl sulfoxide (DMSO) to enhance the miscibility with polymeric solution. After 2 h, the solutions were mixed and left at room temperature (25 °C) without stirring. The precipitation of microparticles was observed after 24 h. Subsequently, the supernatant was removed by decantation, the solid was washed with three portions of fresh THF and dried at room temperature under vacuum. The prepared MPs contained 10 µg extracts/1 mg MPs or 42 µg extracts/1 mg MPs, respectively. The prepared blank and microparticles loaded with S. cadmica extracts were analyzed by scanning electron microscope (SEM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FTIR), as described previously 10 .
Encapsulation efficiency of extracts. The amount of encapsulated extracts was determined by the UV/ Vis method. 10 mg of the sc-PLA-loaded with S. cadmica extract was dissolved in 10 mL of DMSO/CHCl 3 solution (2:5). DMSO was used to destroy the H-bond between the helix of PLA enantiomers to complete dissolution of sc-PLA microparticles and release all encapsulated and physically absorbed on the surface extract to the solution. After that, the UV/Vis measurement was performed at the wavelength of 324 nm. The amount of extracts was calculated from the calibration curve (Fig. S8). The measurement can be performed without separation of extract from the polymer due to the inability of absorption of PLA in the region from 280 to 750 nm. In vitro biocompatibility. Cell cultures and stimulation conditions. The L929 mouse fibroblasts (purchased in LGC Standards, Middlesex, UK), Cavia porcellus (guinea pig) primary gastric epithelial cells isolated from gastric tissue, guinea pig fibroblasts CRL-1405 ATTC (purchased in American Type Culture Collection, Rockville, Manassas, VA, USA), and human THP-1 monocytes (purchased in ATCC TIB-202) or modified THP1-X Blue cells (purchased in Invitrogen, San Diego, CA, USA), were used in this study. Cells were cultured and passaged as previously described [16][17][18]76 . Human THP-1 cells were grown in Roswell Park Memorial Institute (RPMI-1640) medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM/mL l-glutamine at 37 °C, (all in Biowest, Nuaillé, France), in a humid atmosphere containing 5% CO 2 .
THP-1XBlue cells were obtained by transfection of THP-1 cells with a reporter plasmid, the expression of which leads to the secretion of alkaline phosphatase (SEAP) under the control of a promoter induced by the transcription factors nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1). Upon stimulation of the cell surface toll-like receptor (TLR), transcription factors are activated and cells secrete SEAP, which can be detected using the commercial QUANTI-Blue reagent (Invitrogen, San Diego, CA, USA).
Cell viability assay. Biocompatibility of S. cadmica extracts (root and aerial part, separately) and stereocomplexed PLA microparticles, empty or loaded with extracts, as described above, was tested using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) reduction assay according to the ISO norm 10993-5  Cell apoptosis. Primary gastric epithelial cells and guinea pig fibroblasts after exposure to S. cadmica extract (root and aerial part) alone, sc-PLA or E. coli LPS (as a positive control) were immunohistochemically stained for the presence of early pro-apoptotic caspase 3 (CC3) or middle apoptosis stage caspase 9 (CC9) as well as late apoptosis protein carbamoyl-phosphate synthetase 2 asparate transcarbamylase, and dihydroorotase (CAD). The procedure of staining was performed using fluorescently labeled rabbit specific primary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), and then anti-rabbit Alexa Fluor 488-IgG secondary antibody (Invitrogen, Waltham, MA, USA), as previously described [16][17][18] . The amount of CC9 and CAD was determined by measuring fluorescence intensity, at 495 nm excitation and 519 nm emission, using a multifunctional SpectraMax i3 reader (Molecular Devices, San Jose, CA, USA). Four independent experiments were carried out in triplicate for each experimental variant. Apoptosis was also determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as previously described [16][17][18] . The cell nuclei were stained using the fluorescent dye DAPI (Sigma-Aldrich, Saint Louis, MO, USA), which has a strong affinity to the AT base pair in DNA, as previously described 14 . Cell nuclei were imaged under a fluorescent microscope (Zeiss, Axio Scope, A1, Jena, Germany), at a wavelength of 358 nm (excitation) and 461 nm (emission), and the percentage of cells with blebbing nuclei was assessed. Four independent experiments were performed in triplicate.
In vivo biocompatibility of tested microparticles. Biocompatibility of tested microparticles was assessed according to norm EN ISO 10993-1 "Biological evaluation of medical devices/Evaluation and testing in the risk management process/Biological evaluation process/Research in biological evaluation". www.nature.com/scientificreports/ Before per os inoculation, with sc-PLA (24 h), guinea pigs received orally 1 mL of 0.2N NaHCO 3 without anesthesia to increase the stomach pH to neutral, and after 5 min 1 mL of tested microparticles: sc-PLA (mmm), sc-PLA-SCAPE (mmm) and sc-PLA-SCRE (mmm) in 0.85% NaCl. Control animals received 1 mL of 0.85% NaCl. Nonpolar microparticles were prepared according to the norm PN EN ISO 10993-12:2012E. After 24 h, 48 and 72 h: (from inoculation with microparticles, the animals were euthanized by administering a lethal dose (overdosage, 100 mg/kg.b.m) of sodium barbiturate (Morbital, Biowet, Puławy, Poland), and then the blood and organs (spleen, liver) were collected and examined to exclude tissue disorders, and used for further testing.
In all animals the proliferative activity of spleen leucocytes was determined towards cells stimulated with phytohemagglutinin (PHA) (Sigma-Aldrich, Saint Louis, MO, USA) as positive control or cells in culture medium alone (spontaneous proliferation). Briefly, splenic cell suspensions in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine and 100 ug/mL penicillin/streptomycin were added to 96-well plate (5 × 10 5 cells/well). PHA was added to the final concentration of 5 µg/mL. After 72 h cultivation, the culture medium was removed, and cells were frozen at − 70 °C until further processing. Proliferation assessment was performed on the basis of incorporation of radioactive thymidine-3 H[dRT] to DNA of dividing cells, as previously described 16 . The concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in liver homogenates and serum samples was determined by the commercial ELISA (MyBiosoure, San Diego, USA), with the sensitivity 0.06 ng/mL and < 0.118 ng/mL, respectively, according to the attached protocol. Furthermore, the level of serum pro-inflammatory cytokines: tumor necrosis factor alfa (TNF-α) and interleukin (IL)-1B was examined by the ELISA (Thermo Fisher Scientific, Waltham, MA, USA), a sensitivity 1.7 pg/mL (TNF α) and 1 pg/mL (IL-1B), respectively, as recommended by the manufacturer. Three independent experiments were performed in triplicate for each experimental variant.
Isolation and stimulation of guinea pig bone marrow macrophages. The guinea pig bone marrow macrophages were isolated to cRPMI medium from tibias and femurs as previously described 78 . Cells were adjusted to the density of 5 × 10 6 cells/mL in cRPMI culture medium and underwent stimulation with H. pylori or S. cadmica extracts. H. pylori reference strain CCUG 17874 (Culture Collection, University of Gothenburg, Gothenburg, Sweden), positive for vacuolating cytotoxin A (VacA) and cytotoxin associated gene A (CagA) protein, was cultured under microaerophilic conditions according to the previously described procedure 16 . Macrophages were stimulated with H. pylori using the multiplicity of infection (MOI): 10:1 while S. cadmica extracts were used at a concentration 1.25 mg/mL. The procedure of macrophage stimulation was as follows: priming with S. cadmica extract for 24 h and 5 days restimulation with the homologous S. cadmica extract; priming with S. cadmica extract for 24 h, 5 days restimulation with H. pylori; priming with S. cadmica extract for 24 h, 5 days restimulation with H. pylori, and additional 24 h restimulation with homologous S. cadmica extract. Stimulated or unstimulated control cells were then examined for phagocytic activity in conjunction with an expression of CD11b activation marker and production of cytokines, TNF-α and IL-10.
Phagocytosis. Bone marrow macrophages (5 × 10 6 cells/mL) were applied to the wells of a 96-well plate (100 µL/well), and stimulated with S. cadmica extract (aerial part or root) or with live H. pylori as described above. Phagocytosis was assessed using fluorescein-labeled Escherichia coli, as recommended by the manufacturer (Vybrant Phagocytosis Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA). Intensity of phagocytosis was determined by measuring the fluorescence using a multifunctional reader SpectraMax i3 (Molecular Devicesat, San Jose, CA, USA) at 495 nm (excitation) and 515 nm (emission). Four independent experiments were carried out in triplicate for each experimental variant.
Surface deposition of CD11b: immunofluorescence. The guinea pig bone marrow macrophages after priming and restimulation as described above, were prepared for staining with fluorescently labeled primary and secondary antibodies as previously described 16 . We used rabbit anti-CD11b antibodies (Thermo Fisher Scientific, Waltham, MA, USA), diluted 1:200 in 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Cells were then treated with the secondary goat anti-rabbit antibody Alexa Fluor488-labeled (Invitrogen, CA USA), diluted 1:200. The intensity of fluorescence was measured using a multifunctional reader SpectraMax i3 (Molecular Devicesat, San Jose, CA, USA) at the wavelengths for Alexa Fluor488 (excitation 495 nm, emission 519 nm). Four independent experiments were carried out in triplicate for each experimental variant.
ELISA for TNF-α and IL-10. After stimulation of macrophages, cell culture supernatants were tested for TNF-α and IL-10 by the commercial ELISA (Thermo Fisher Scientific, Waltham, MA, USA), with the sensitivity 1.7 pg/ mL (TNF-α) and 1 pg/mL (IL-10) on the basis of standard curves for these cytokines, as recommended by the manufacturer. Four independent experiments were carried out in triplicate for each experimental variant. Statistical analysis. Data were expressed as median values ± range. The differences between groups were tested using the non-parametric Mann-Whitney U test or Kruskal-Wallis test. For statistical analysis the Statistica 13 PL software (https:// stati stica. softw are. infor mer. com/ 13. 3soft ware) (Kraków, Poland) was used. Results were considered statistically significant when p < 0.05.

Data availability
All data generated or analyzed during this study are included in this published article or Supplementary File.